Identification of a nuclear protein that constitutively recognizes the sequence containing a heat‐shock element: Its binding properties and possible function modulating heat‐shock induction of the rat heme oxygenase gene

Heme oxygenase is an essential enzyme in heme catabolism, and also known as a 32‐kDa heat‐shock protein in rat. The rat heme‐oxygenase gene promoter contains a functional heat‐shock element (HSE) designated as HSE1 (−290 to −276 from the transcriptional initiation site), which consists of three copies of a 5‐bp unit (5′‐NGAAN‐3′; →) in alternating orientation. Here we identified a putative HSE (−221 to −212), designated as HSE2, consisting of an inverted repeat of this 5‐bp unit (←→). Using transient expression assays, we show that HSE1 is sufficient to confer the heat‐inducibility (a three fold to fourfold increase) on the reporter gene located downstream from the rat heme‐oxygenase gene promoter, but HSE2 alone is not, suggesting that HSE2, a HSE of a tail‐to‐tail configuration, is not functional in vivo. However, the presence of both HSE1 and HSE2 in the promoter region increased the heat‐mediated induction of the reporter‐gene expression by more than 15‐fold, Gel mobility‐shift assays indicate that both HSE1 and HSE2 are recognized by activated heat‐shock factor present only in heat‐shocked rat glioma cells. Interestingly, the sequence containing HSE2 is also bound by a protein that is present in nuclear extracts prepared from either heat‐shocked or non‐shocked glioma cells, but this nuclear protein is nuable to bind to HSE1. We suggest that a protein binding to the sequence containing HSE2 may be involved in transcriptional regulation of the rat heme oxygenase gene under thermal stress.